Lipoprotein-associated coagulation inhibitor (LACI) is a protein inhibitor present in mammalian blood plasma. LACI is also known as tissue factor (TF) inhibitor, tissue thromboplastin (Factor III) inhibitor, extrinsic pathway inhibitor (EPI) and tissue factor pathway inhibitor (TFPI).
Blood coagulation is the conversion of fluid blood to a solid gel or clot. The main event is the conversion of soluble fibrinogen to insoluble strands of fibrin, although fibrin itself forms only 0.15% of the total blood clot. This conversion is the last step in a complex enzyme cascade. The components (factors) are present as zymogens, inactive precursors of proteolytic enzymes, which are converted into active enzymes by proteolytic cleavage at specific sites. Activation of a small amount of one factor catalyses the formation of larger amounts of the next, and so on, giving an amplification which results in an extremely rapid formation of fibrin.
The coagulation cascade which occurs in mammalian blood is divided by in vitro methods into an intrinsic system (all factors present in the blood) and an extrinsic system which depends on the addition of thromboplastin. The intrinsic pathway commences when the first zymogen, factor XII or `Hageman Factor`, adheres to a negatively charged surface and in the presence of high molecular weight kininogen and prekallikren, becomes an active enzyme, designated XIIa. The activating surface may be collagen which is exposed by tissue injury. Factor XIIa activates factor XI to give XIa, factor XIa activates factor IX to IXa and this, in the presence of calcium ions, a negatively charged phospholipid surface and factor VIIIa, activates factor X. The negatively charged phospholipid surface is provided by platelets and in vivo this serves to localize the process of coagulation to sites of platelet deposition. Factor Xa, in the presence of calcium ions, a platelet-derived negatively charged phospholipid surface and a binding protein, factor V, activates prothrombin to give thrombin (IIa)--the main enzyme of the cascade. Thrombin, acting on gly-arg bonds, removes small fibrinopeptides from the N-terminal regions of the large dimeric fibrinogen molecules, enabling them to polymerize to form strands of fibrin. Thrombin also activates the fibrin stabilizing factor, factor XIII, to give XIIa, a fibrinoligase, which, in the presence of calcium ions strengthens the fibrin-to-fibrin links with intermolecular .gamma.-glutamyl-.epsilon.-lysine bridges. In addition, thrombin acts directly on platelets to cause aggregation, and release of subcellular constituents and arachidonic acid. A further function of thrombin is to activate the coagulation inhibitor, protein C. Factors XIIa, XIa, IXa, Xa, and thrombin are all serine proteases.
The extrinsic pathway in vivo is initiated by a substance generated by, or exposed by, tissue damage and termed `tissue factor`, interacting with Factor VII in the presence of calcium ions and phospholipid to activate factors X and IX, after which the sequence proceeds as already described. The identity of TF is known. There is evidence that tissue factor occurs in the plasma membranes of perturbed endothelial cells of blood vessels and also in atheromatous plaques.
The two pathways described are not entirely separate because both factor IXa and factor XIIa in the intrinsic pathway may activate factor VII in the extrinsic pathway. There are, in addition, various feedback loops between other factors, which enhance reaction rates. For example, thrombin (IIa) enhances the activation of both factor V and factor VIII.
Sepsis and its sequela septic shock remain among the most dreaded complications after surgery and in critically ill patients. The Center for Disease Control ranks septicemia as the 13th leading cause of death in the United States (see MMWR, 1987, 39:31 and US Dept. of Health and Human Services, 37:7, 1989), and the 10th leading cause of death among elderly Americans (see MMWR, 1987, 32:777). The incidence of these disorders is increasing, and mortality remains high. Estimates of the total cost of caring for patients with septicemia range from $5 billion to $10 billion annually (see MMWR, 1987, 39:31). Death can occur in 40% to 60% of the patients. This percentage has not seen any improvement over the past 20 years. The incidence of blood borne gram-positive and gram-negative infections that can lead to septic shock occur approximately equally.
Sepsis is a toxic condition resulting from the spread of bacteria, or their products (collectively referred to herein as bacterial endotoxins) from a focus of infection. Septicemia is a form of sepsis, and more particularly is a toxic condition resulting from invasion of the blood stream by bacterial endotoxins from a focus of infection. Sepsis can cause shock in many ways, some related to the primary focus of infection and some related to the systemic effects of the bacterial endotoxins. For example, in septacemia, bacterial endotoxins, along with other cell-derived materials, such as IL 1, IL-6 and TNF, activate the coagulation system and initiate platelet aggregation. The process leads to blood clotting, a drop in blood pressure and finally kidney, heart and lung failure.
Septic shock is characterized by inadequate tissue perfusion, leading to insufficient oxygen supply to tissues, hypotension and oliguria. Septic shock occurs because bacterial products, principally LPS, react with cell membranes and components of the coagulation, complement, fibrinolytic, bradykinin and immune systems to activate coagulation, injure cells and alter blood flow, especially in the microvasculature. Microorganisms frequently activate the classic complement pathway, and endotoxin activates the alternate pathway. Complement activation, leukotriene generation and the direct effects of endotoxin on neutrophils lead to accumulation of these inflammatory cells in the lungs, release of the enzymes and production of toxic oxygen radicals which damage the pulmonary endothelium and initiate the acute respiratory distress syndrome (ARDS). ARDS is a major cause of death in patients with septic shock and is characterized by pulmonary congestion, granulocyte aggregation, hemorrhage and capillary thrombi.
Activation of the coagulation cascade by bacterial endotoxins introduced directly into the bloodstream can result in extensive fibrin deposition on arterial surfaces with depletion of fibrinogen, prothrombin, factors V and VIII, and platelets. In addition, the fibrinolytic system is stimulated, resulting in further formation of fibrin degradation products. Disseminated intravascular coagulation (DIC) is a complex coagulation disorder resulting from widespread activation of the clotting mechanism or coagulation cascade which, in turn, results from septicemia. Essentially, the process represents conversion of plasma to serum within the circulation system. Such process represents one of the most serious acquired coagulation disorders. Some common complications of disseminated intravascular coagulation are severe clinical bleeding, thrombosis, tissue ischaemia and necrosis, hemolysis and organ failure.
At the same time, as coagulation is apparently initiated by endotoxin, countervening mechanisms also appear to be activated by clotting, namely activation of the fibrinolytic system. Activated Factor XIII converts plasminogen pro-activator to plasminogen activator which subsequently converts plasminogen to plasmin thereby mediating clot lysis. The activation of plasma fibrinolytic systems may therefore also contribute to bleeding tendencies.
Endotoxemia is associated with an increase in the circulating levels of tissue plasminogen activator inhibitor (PAI). This inhibitor rapidly inactivates tissue plasminogen activator (TPA), thereby hindering its ability to promote fibrinolysis through activation of plasminogen to plasmin. Impairment of fibrinolysis may cause fibrin deposition in blood vessels, thus contributing to the disseminated intravascular coagulation associated with septic shock.
Disseminated intravascular coagulation (DIC) is a coagulopathic disorder that occurs in response to invading microorganisms characterized by widespread deposition of fibrin in small vessels. The initiating cause of DIC appears to be the release of thromboplastin (tissue factor) into the circulation. During this process, there is a reduction in fibrinogen and platelets, and a rise in fibrin split products resulting in fibrin deposition in blood vessels. The sequence of events that occur during DIC are described in FIG. 1. The patients either suffer from thrombosis or hemorrhage depending on the extent of exhaustion of the coagulation protease inhibitors during the disease process. Part of the regulation of the coagulation cascade depends on the rate of blood flow. When flow is decreased, as it is in DIC and sepsis, the problems are magnified. DIC (clinically mild to severe form) is thought to occur with high frequency in septic shock patients and several other syndromes such as head trauma and burns, obstetric complications, transfusion reactions, and cancer. A recent abstract by Xoma Corporation indicates that DIC was present on entry in 24% of septic patients (Martin et al., 1989, Natural History in the 1980s, Abstract No. 317, ICAAC Meeting, Dallas). Furthermore, the abstract describes that DIC and acute respiratory distress syndrome were the variables most predictive of death by day 7 (risk ratios 4 and 2.3). The cascade of events that lead to release of tissue factor into circulation and sepsis is very complex. Various cytokines are released from activated monocytes, endothelial cells and others; these cytokines include tumor necrosis factor (TNF), interleukin 1 (IL-1) (which are known to up-regulate tissue factor expression), interleukin 6 (IL-6), gamma interferon (IFN-.gamma.), interleukin 8 (IL-8), and others. The complement cascade is also activated as demonstrated by the rise in C3a and C5a levels in plasma of septic patients. Consequently, an agent that will treat coagulation without affecting the expression of tissue factor or its activity will not necessarily be effective to treat sepsis.
There are currently no satisfactory interventions for the prevention or treatment of sepsis or DIC. Heparin is the most commonly used anticoagulant in DIC. However, it has been controversial because it can induce bleeding and worsen the patient's condition. See, for example, Corrigan et al., "Heparin Therapy in Septacemia with Disseminated Intravascular Coagulation. Effect on Mortality and on Correction of Hemostatic Defects", N. Engl J. Med., 283:778-782 (1970); Lasch et al., Heparin Therapy of Diffuse Intravascular Coagulation (DIC)", Thrombos. Diathes. Haemorrh., 33:105 (1974); Straub, "A Case Against Heparin Therapy of Intravascular Coagulation", Thrombos. Diathes. Haemorrh., 33:107 (1974).
Other attempts to treat sepsis using an anticoagulant have also been difficult. As shown in Taylor et al., 1991, Blood, 78:364-368, warfarin and heparin are mentioned as two anticoagulants that are used to treat DIC in sepsis, but neither are the ideal drugs. Additionally, Taylor et al. show that a new drug DEGR-Xa, a factor Xa antagonist, can inhibit DIC, however, this drug failed to block the lethal effects of sepsis. Consequently, it is evident that an agent which may interrupt the coagulation pathway is not necessarily effective as an inhibitor of septic shock. Therefore, there is a need in the art for a composition that will inhibit the lethal effects of sepsis.